Method for identifying duck palm weight and application of related molecular marker

Abstract

The application discloses a method for identifying duck palm weight and a related molecular marker application, relates to the technical field of molecular marker assisted selection, and a primer pair is used for amplifying a DNA fragment containing a polymorphic site of a duck reference genome IASCAAS_PekinDuck_T2T chromosome 4 from the 5' end 63823029 base position; the duck reference genome IASCAAS_PekinDuck_T2T is a duck reference genome sequence in a GenBank database; the duck is a Zhongxinbaihu meat duck, and the primer pair consists of a DNA molecule shown in SEQ ID No. 2 and a DNA molecule shown in SEQ ID No. 3. The method for identifying the duck palm weight and the related molecular marker application can determine the genotype of the duck by detecting the genomic DNA of the duck, realize early in-vivo screening of the duck palm weight trait, do not need to wait for post-slaughter determination of a phenotype, significantly shorten a breeding cycle, and reduce breeding cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular marker assisted selection, in particular to a method for identifying duck palm weight and related molecular marker application thereof. BACKGROUND

[0002] With the upgrading of catering consumption and the continuous expansion of the segmented meat market, consumers' demand for duck meat products has shifted from simply pursuing overall output to focusing on the specifications and quality of specific parts (such as duck palms). In meat duck production, duck palm weight is an important slaughter trait, directly affecting individual dressing percentage and the economic value of segmented products. Duck palms with greater weight are more favored by the catering industry and food processing enterprises, which can bring higher economic benefits and market competitiveness to breeding enterprises.

[0003] From the perspective of duck physiology, duck palms not only bear the role of supporting the carcass, walking, and turning in water, but also affect mating behavior. Therefore, breeding for duck palm-related traits can indirectly improve the growth performance and reproductive efficiency of ducks. Currently, the breeding process for meat duck slaughter-related traits is accelerating, and duck palms, as high-value parts, are gradually gaining attention from researchers and breeders, and are expected to be included in mainstream meat duck breeding programs.

[0004] In traditional breeding, the selection of duck palm weight relies on post-mortem phenotypic determination, which cannot achieve early screening in vivo, resulting in long breeding cycles, high costs, and low selection efficiency. Therefore, it is crucial to identify key molecular markers affecting duck palm weight and develop their applications. SUMMARY

[0005] The present application aims to provide a method for identifying duck palm weight and related molecular marker application thereof, to achieve early screening of duck palm weight traits in vivo, shorten the breeding cycle, and improve selection efficiency and economic benefits.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solution: a method for identifying duck palm weight, the primer pair is used to amplify a DNA fragment containing a polymorphic site at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T; the duck reference genome IASCAAS_PekinDuck_T2T is the duck reference genome sequence in the GenBank database; and the duck is a Zhongxinbaihu meat duck.

[0007] Further, the primer pair consists of a DNA molecule represented by SEQ ID No. 2 and a DNA molecule represented by SEQ ID No. 3.

[0008] Further, the method includes the following content: the duck palm weight of ducks with TT genotype and CT genotype is significantly higher than that of ducks with CC genotype (P < 0.05);

[0009] The duck of the TT genotype is a duck in which the base at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T is T;

[0010] The duck of the CT genotype is a duck in which the bases at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T are C and T;

[0011] The duck of the CC genotype is a duck in which the base at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T is C;

[0012] The duck reference genome IASCAAS_PekinDuck_T2T is a duck reference genome sequence in the GenBank database; and the duck is a Zhongxinbaihu meat duck.

[0013] Further, the determination method of the TT genotype, the CT genotype or the CC genotype is as follows: taking the genomic DNA of the duck as a template, performing PCR amplification with the primer pair in claim 1 or 2 as primers to obtain a PCR amplification product; if the base at position 207 from the 5' end of the PCR amplification product is T, then the genotype of the duck is the TT genotype; if the base at position 207 from the 5' end of the PCR amplification product is C and T, then the genotype of the duck is the CT genotype; and if the base at position 207 from the 5' end of the PCR amplification product is C, then the genotype of the duck is the CC genotype.

[0014] Further, the method is breeding the duck of the TT genotype;

[0015] The duck of the TT genotype is a duck in which the base at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T is T;

[0016] The duck reference genome IASCAAS_PekinDuck_T2T is a duck reference genome sequence in the GenBank database; and the duck is a Zhongxinbaihu meat duck.

[0017] Further, the determination method of the TT genotype is as follows: taking the genomic DNA of the duck as a template, performing PCR amplification with the primer pair in claim 1 or 2 as primers to obtain a PCR amplification product; and if the base at position 207 from the 5' end of the PCR amplification product is T, then the genotype of the duck is the TT genotype.

[0018] Further, the method is breeding the duck of the CC genotype;

[0019] The duck of the CC genotype is a duck whose base at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T is C;

[0020] The duck reference genome IASCAAS_PekinDuck_T2T is a duck reference genome sequence in the GenBank database; and the duck is a Zhongxinbaihuameidajia.

[0021] Further, the determination method of the CC genotype is as follows: taking the genomic DNA of the duck as a template, and taking the primer pair in claim 1 or 2 as primers to perform PCR amplification to obtain a PCR amplification product; if the base at position 207 from the 5' end of the PCR amplification product is C, then the genotype of the duck is the CC genotype.

[0022] Further, a related molecular marker application is applied to the method for identifying the duck foot weight.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The method for identifying the duck foot weight and the related molecular marker application thereof can determine the genotype of the duck by detecting the genomic DNA of the duck, realize early in-vivo screening of the duck foot weight trait, do not need to wait for post-slaughter determination of the phenotype, significantly shorten the breeding cycle, and reduce the breeding cost;

[0025] The molecular marker of the present application is significantly related to the duck foot weight, the duck foot weight corresponding to different genotypes has clear differences, and the selection accuracy of the duck foot weight trait can be significantly improved by assisted selection through the marker;

[0026] The primer pair used for detection has high specificity, the PCR amplification condition is mild, the sequencing or genotype detection method is mature, batch detection can be realized, the detection cost is low, and the method is suitable for screening of large-scale breeding populations.

[0027] Not only suitable for Zhongxinbaihuameidajia, but also can be popularized to other duck breeds, and provides a universal technical means for genetic improvement of the duck foot weight trait, and has very high industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Fig. 1 It is a duck foot weight whole genome association analysis result graph of the present application;

[0029] Fig. 2 It is a duck foot weight phenotype graph of g.63823029C>T different genotypes of chromosome 4 of the present application;

[0030] Fig. 3Figure 1 is a sequence of the g.63823029C>T polymorphic site near the fourth chromosome of the reference genome IASCAAS_PekinDuck_T2T of the duck individual of CC, CT, TT genotype according to the present application. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0032] Please refer to Figs. 1-3 The present application provides a technical solution: a method for identifying duck palm weight and application of related molecular markers.

[0033] Embodiment 1: identifying the genotype of duck individual and predicting duck palm weight

[0034] Sample collection: 10 medium and new white-feathered meat ducks to be detected (21 days old, not slaughtered) were selected, 2 mL of wing vein blood of each duck was collected using an EDTA anticoagulant blood collection tube, and was stored at -20℃ for standby.

[0035] Genomic DNA extraction: genomic DNA of each duck was extracted using a Tian Gen blood genomic DNA extraction kit (Cat. No. DP348) according to the kit instruction steps, the concentration and purity of the DNA were detected using a Nanodrop 2000 / 2000C, and it was ensured that the OD260 / OD280 was between 1.8-2.0 and the concentration was ≥50 ng / μL.

[0036] PCR amplification: the genomic DNA of the 10 ducks extracted was used as a template, and the primer pair (SEQ ID No. 2 and SEQ ID No. 3) of the present application was used for PCR amplification.

[0037] The PCR amplification product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for Sanger sequencing, and the sequencing results were analyzed using Chromas software, focusing on checking the type of the 207th base from the 5' end of the amplification product, and determining the genotype of each duck:

[0038] No. 1-3: the 207th base is T→genotype TT;

[0039] No. 4-7: the 207th base is C / T→genotype CT;

[0040] No. 8-10: the 207th base is C→genotype CC.

[0041] According to the genotype prediction, the duck palm weight of the duck with TT and CT genotype is greater than that of the duck with CC genotype. After 10 ducks are raised to 42 days old, they are uniformly slaughtered to determine the actual duck palm weight:

[0042] No. 1-3: the actual average duck palm weight is 74.6g;

[0043] No. 4-7: the actual average duck palm weight is 72.4g;

[0044] No. 8-10: the actual average duck palm weight is 69.9g.

[0045] Based on the actual duck palm weight, the duck palm weight of the duck with TT and CT genotype is greater than that of the duck with CC genotype, which is consistent with the above verification and prediction results.

[0046] The verification results show that the method of the present application can accurately predict the duck palm weight.

[0047] Example 2: Breeding of medium-sized new white-feather meat duck with larger palm weight by using molecular marker

[0048] The breeding population is selected from the basic breeding population (a total of 500, 20 days old, half male and half female) of L4 line of medium-sized new white-feather meat duck.

[0049] The genotype detection is performed according to the method of Example 1, the wing vein blood of each duck is collected, the genomic DNA is extracted, the PCR amplification and sequencing are performed, and the genotype of each duck is determined:

[0050] TT genotype: 120 (62 females and 58 males);

[0051] CT genotype: 250 (128 females and 122 males);

[0052] CC genotype: 130 (65 females and 65 males).

[0053] The breeding selection preferentially selects the duck with TT genotype as the core breeding population (the breeding rate is 100%), and simultaneously selects the individual with excellent growth performance from the duck with CT genotype (the breeding rate is 30%) as the auxiliary breeding population, and eliminates all individuals with CC genotype. Finally, the core breeding population of 120 (TT) + auxiliary breeding population of 75 (CT) is determined, with a total of 195.

[0054] 200 individuals of the breeding offspring are selected, raised to 42 days old, and slaughtered to determine the duck palm weight, and the duck palm weight of 200 individuals of the control group (the same period breeding population without genotype selection) is determined:

[0055] The average duck palm weight of the offspring of the breeding population selected by genotype is 73.2g;

[0056] The average duck palm weight of the control group is 70.9g;

[0057] The average duck palm weight of the offspring of the breeding population selected by genotypes is increased by 2.3 g, and the difference is significant (P < 0.05).

[0058] The results show that the duck palm weight of the offspring population can be significantly improved by the molecular marker assisted selection of the application.

[0059] It should be noted that in this paper, the term "including", "containing" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or equipment.

[0060] The principles and implementation modes of the application are described by applying specific examples in this paper, and the above examples are only used to help understand the method of the application and its core idea. The above description is only the preferred embodiment of the application, and it should be pointed out that due to the limitation of language expression, there are infinite specific structures, and for ordinary skilled persons in the technical field, some improvements, decorations or changes can be made without departing from the principles of the application, and the above technical features can be combined in a proper way; the improvements, decorations, changes or combinations, or the direct application of the inventive concept and technical scheme to other occasions without improvement, shall be regarded as the protection scope of the application.

Claims

1. A method for determining the weight of the feet of a new type of white-feathered meat duck, characterized in that: A DNA fragment containing the polymorphic site at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T was amplified; the duck reference genome IASCAAS_PekinDuck_T2T is a duck reference genome sequence from the GenBank database; the method includes the following: The foot weight of ducks with TT and CT genotypes was significantly higher than that of ducks with CC genotype (P < 0.05). The ducks with the TT genotype are those whose bases on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T are T, located at position 63823029 from the 5' end. The ducks with the CT genotype are those whose bases at position 63823029 from the 5' end of chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T are C and T. The CC genotype ducks are those whose base position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T is C.

2. An application of a molecular marker related to duck foot weight, characterized in that: A DNA fragment containing the polymorphic site at position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T was amplified; the duck reference genome IASCAAS_PekinDuck_T2T is a duck reference genome sequence from the GenBank database; the method includes the following: The foot weight of ducks with TT and CT genotypes was significantly higher than that of ducks with CC genotype (P < 0.05). The ducks with the TT genotype are those whose bases on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T are T, located at position 63823029 from the 5' end. The ducks with the CT genotype are those whose bases at position 63823029 from the 5' end of chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T are C and T. The CC genotype ducks are those whose base position 63823029 from the 5' end on chromosome 4 of the duck reference genome IASCAAS_PekinDuck_T2T is C.

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